Plasmids used in this study.

<p>All plasmids are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a>. Large arrowheads represent the terminal sequences of <i>piggyBac</i>. Act5C, promoter from <i>D. melanogaster</i> gene <i>Actin5C; Hygromycin^R,</i> coding region for bacterial gene <i>hygromycin B phosphotransferase</i>; ie1, promoter from the baculovirus gene <i>immediate early 1</i>; Amp^R, bacterial gene <i>beta-lactamase</i>; hsp70, promoter from <i>D. melanogaster</i> gene <i>hsp70</i>; PB-transposase, coding region for <i>piggyBac</i> transposase; DsRed, coding region for <i>Discosoma sp</i>. gene <i>red fluorescent protein</i>; pUb, promoter from <i>D. melanogaster</i> gene <i>pUbi-p63e</i>; Kan^R, bacterial gene <i>Neomycin phosphotransferase II</i>; gDNA, refers to <i>Aedes aegypti</i> genomic DNA flanking the 5′ and 3 ends of <i>piggyBac</i> elements integrated in the genome of cell line AagPB8 (in pCL1w+) and in transgenic line 40D (in p40Dw+; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#pone.0068454-Sethuraman1" target="_blank">[25]</a>); mini-white, the <i>D. melanogaster</i> gene <i>w^+mW.hs;</i> attB, the bacterial attachment site for phage <i>ΦC31.</i></p

Location of <i>piggyBac</i> integration sites in AegPB8.

*<p>Position of underlined nucleotide shown based on <i>Aedes aegypti</i> genome version 66.1 (AegL1)</p

Plasmid-based <i>piggyBac</i> excision assay.

<p>A) Diagrammatic representation of the <i>piggyBac</i>-containing plasmid, <i>piggyBac</i> 3×P3EGFP used in the excision assay described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a> (donor plasmid), and the same plasmid following precise excision of the <i>piggyBac</i> element (excision plasmid). The <i>piggyBac</i>-containing donor plasmid, <i>piggyBac</i> 3×P3EGFP, and <i>piggyBac</i> transposase expressing helper plasmid, pHspPBtpase:PubDsRed, were co-transfected into AagPB8 cells. Transfected cells were heat-shocked after 12 hrs and collected after 72 hrs. DNA was extracted and used as a template for PCR. Primers 1 and 2 (shown as labeled short half-arrows) were specific to the donor-plasmid backbone (494donorFWD, 494donorREV) and yield a 751 bp product (grey line) in the presence of the donor and excision plasmids. Primers 3 and 4 (494excisionFWD, 494excisionREV and shown as labeled short half-arrows) are specific to the plasmid DNA flanking the <i>piggyBac</i> element, however under the conditions of this experiment PCR products were only detected if donor plasmids missing the <i>piggyBac</i> element through excision were present, yielding a 540 bp PCR product (grey line). The 5′ and 3′ terminal <i>piggyBac</i> sequences are represented by arrows (5′PB, 3′PB). The duplicated TTAA target sequence into which <i>piggyBac</i> integrated is shown as a black diamond and the 3×P3EGFP transgene within the <i>piggyBac</i> element is shown as a black rectangle. The normally circular plasmids are represented as linear molecules. B) The PCR results from two <i>piggyBac</i> excision assays in AagPB8 cells. Lanes 1 and 2: from cells transfected with donor and <i>pHspPBtpase:PubDsRed</i> (2 independent transfections). Lane 3: from cells transfected with donor and control plasmids (pBluescript SKII+). Lane 4 and 5: positive controls for detecting excision events. The DNA used as a template in these reactions was a purified excision plasmid recovered from a previous excision assay (2 independent transfections). Lane 6: negative control for detecting excision events. DNA used as a template in this reaction came from cells transfected with donor plasmid only, without the transposase helper plasmid. Two PCR reactions were performed on each sample using primer combinations indicated above the lanes numbers. Primers 1+2 (same primers referred to in panel A) detected the presence of donor and excision plasmids and yielded a 751 bp reaction product (white arrow). Primers 3+4 (same primers referred to in panel A) yielded a 540 bp reaction product (white arrow) only when the <i>piggyBac</i> element in the donor plasmid had excised. Only the 540 and 751 bp bands are specific reaction products.</p

<i>piggyBac</i> transposable element display results using DNA isolated from cell line AagPB8.

<p>Lanes 1 are the results using DNA as a template isolated from cell line AagPB8 and shows evidence of the 5′ end of one of the two <i>piggyBac</i> elements that had integrated by canonical cut-and-paste transposition –80 bp band. The 5′ end of the second <i>piggyBac</i> element that integrated by canonical cut-and-paste transposition is not visible. This element can be detected when the 3′ ends of integrated <i>piggyBac</i> elements are visualized using transposable element display (not shown). The band at 250 bp is the <i>piggyBac</i> element associated with a copy of the integrated plasmid pBac:Act5cHyg:ie1EGFP. The sample was loaded into two adjacent lanes. Lanes 2 are the results using DNA as a template isolated from non-transgenic Aag-2 cells and this serves as a negative control for this assay since there are no <i>piggyBac</i> elements in <i>Ae. aegypti</i>. The sample was loaded into two adjacent lanes. Lanes 3 are the results using DNA as a template from AagPB8 cells 72 hours after being transfected with <i>piggyBac</i>-transposase-expressing pHspPBtpase:PubDsRed. The sample was loaded into two adjacent lanes. There was no evidence of <i>piggyBac</i> elements in other positions in the genome in Lane 3 as would be expected if <i>piggyBac</i> transposase mobilized the integrated <i>piggyBac</i> elements in AagPB8 cells. The asterisk indicated the position of a non-specific TE display band present in all samples. The positions of molecular weight markers 80 bp and 250 bp in length are shown.</p

Phylogenetic relationships among Gypsy retrotransposons inferred from Neighbor-Joining analysis of RT/RH amino acid sequences.

<p>The crustacean elements are indicated in bold and the four <i>R. exoculata</i> elements (GyRex) are highlighted in grey. Statistical support (>50%) comes from non parametric bootstrapping using 100 replicates. DIRS1-like sequences were used as outgroup.</p

Number of Copia and Gypsy elements studied in crustaceans.

<p>Genetic relationships between crustacean classes and orders are represented by a tree topology reconstructed from previous studies (Regier <i>et al.</i> 2010, Giribet and Edgecombe, 2011; Ahyong and O’Meally, 2004). <b>M</b>: Malacostraca, <b>D</b>: Decapoda. For Copia retrotransposons, GalEa and non-GalEa elements are distinguished. Only a few representatives of the Copia elements described <i>in D. pulex</i> were studied. nt: not tested; -: no element detected; ^a species screened using degenerate PCRs.</p

Phylogenetic relationships among Copia retrotransposons inferred from Neighbor-Joining analysis of RT/RH amino acid sequences.

<p>The crustacean elements are indicated in bold and the three <i>R. exoculata</i> elements (CoRex) are highlighted in grey. Statistical support (>50%) comes from non parametric bootstrapping using 100 replicates. Gypsy sequences were used as outgroup.</p

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes

<div><p>Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of <i>Schizosaccharomyces pombe</i> is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 <i>S</i>. <i>pombe</i> strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in <i>Saccharomyces cerevisiae</i>, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.</p></div

Host factors that function in Tf1 integration.

<p>Host factors that function in Tf1 integration.</p

The quantitative homologous recombination assay detected deletion strains with reduced recombination that was not detected with the patch assay.

<p>A. Quantitative recombination assays of deletion strains expressing wild-type Tf1-<i>nat</i>AI and the INfs. B. Quantitative recombination frequencies are shown in a histogram of strains sorted from highest to lowest. The numbers on the x-axis identify strains in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006775#pgen.1006775.s011" target="_blank">S3 Table</a>. The deletion strains here were shown by the yeast patch assays to have defects in transposition but not homologous recombination (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006775#pgen.1006775.s011" target="_blank">S3 Table</a>). The red line illustrates the homologous recombination activity of wild-type Tf1 in wild-type <i>S</i>. <i>pombe</i>. The green line shows the homologous recombination activity of the INfs in Wild-type <i>S</i>. <i>pombe</i>. C. Quantitative homologous recombination assays of cells with catalytically inactive mutants in the catalytic core (CC) of IN.</p